Rods, tori, and honeycombs: the directed self-assembly of microtissues with prescribed microscale geometries

It is thought that, due to energy and surface area:volume minimization, the spheroid is the terminal structure of cellular self-assembly. We investigated whether self-assembly could be directed to generate complex-shaped structures. Using micromolded, nonadhesive agarose hydrogels seeded with rat hepatoma (H35s), human fibroblasts (NHFs), or their mix (1:1), we show that cells can self-assemble rods, tori, and honeycombs. We found that in trough-shaped recesses up to 2.2 mm long, H35s readily formed rod-like structures stable at 49% the recess lengths. They also formed intact tori (88%) and fully intact honeycombs structures with patent lumens (9/9) even when released from the mold. In contrast, NHFs in trough features progressed rapidly to spheroids and formed fewer stable tori (30%) and honeycombs (0/9). The 1:1 mix of cells self-assembled rapidly like NHFs but were able to form more stable structures (tori: 30%, honeycombs: 3/9). Experiments with labeled cells in tori and honeycombs revealed that cells self-segregated in these complex structures, with H35s enveloping NHFs, and that NHFs had different morphologies in taut vs. relaxed structures. These data open new possibilities for in vitro tissue models for embryo- and organogenesis study as well as for tissue engineering applications.